Abstract To capture the normal brain functions, it is critically important to record the neural activities in freely-behaving animals, with high resolution, high speed, and high throughput. So far, our knowledge about neuronal activity of awake animals mainly relies on electrode recording, which, however, is invasive. Optical imaging techniques have been widely used to visualize activity of a large number of neurons in mouse models using fluorescent membrane voltage or calcium indicators. However, limited by the penetration depth (<1 mm), it is technically challenging to record the brain functions at depths beyond the cortex layer, such as in the hippocampus. A new large-scale recording technology with high resolution and deep penetration in freely-behaving animals would be of great utility for the neuroscience community. Photoacoustic microscopy (PAM) is a promising candidate for this task due to the relatively deep penetration of ultrasound waves. However, PAM has not been able to image neural activities of freely-moving animals, because (1) it is challenging to miniaturize the imaging system, (2) there lacks calcium or voltage probes that can report neural activities in deep brain, and (3) photoacoustic detection sensitivity of molecular probes is traditionally low due to the strong background signals from blood. In this proposal, we plan to overcome all of the above technical obstacles and develop head-mounted photoacoustic imaging of deep-brain neural activities in freely-behaving animals. To achieve this goal, we will follow a three-aim strategy. (1) In Aim 1, we will develop a miniaturized head-mounted PAM (HM-PAM) system. Several key innovations will reduce the system footprint to 1 cm3. HM-PAM will achieve a penetration depth of ~3.0 mm with ~10?15 m resolution, which is deeper than that with pure optical microscopy. (2) In Aim 2, we will develop novel near-infrared photoswitchable genetically-encoded calcium indicators (NIR-PS-GECIs) as PA probes. We will engineer and optimize a new class of NIR-PS-GECIs based on photoacoustic Frster resonance energy transfer (FRET-PA). We have proven that the photoswitching, which enables differential PA imaging, is currently one of the most effective approaches to enhance the PA detection sensitivity. We will thus apply fast photoswitching of the NIR-PS-GEICs to enhance the detection sensitivity of HM-PAM. (3) In Aim 3, the optimized HM-PAM and advanced NIR-PS-GECIs will be thoroughly characterized and validated in dissociated neurons and in vivo. We will perform proof-of-concept experiments of deep-brain neural activity in freely-behaving animals. In summary, our proposal will build on the innovations of the first head-mounted PAM system, the first NIR photoswitching GECIs, and the differential FRET- PA imaging that rejects the strong background blood signals. This enabling technology will provide a powerful toolkit for studying neural activities in health, disease, and behavioral states.